1. Field of the Invention
The present invention relates to a reagent composition for the quantitative analysis of inorganic phosphorus or phosphate contained in a sample and a dry analysis element including a reagent layer containing such a reagent composition.
2. Prior Art Statement
The quantitative analysis of inorganic phosphorus contained in blood serum or urine is an effective clinical chemical test for finding the mineral or electrolyte metabolism, similar to the analysis of calcium in blood serum, and thus it is recognized as one of the important clinical tests for the examination of disorder in kidney function, vitamin D deficiency, acromegaly, hyperparathyroidism or hypoparathyroidism.
The quantitative analysis of inorganic phosphorus, which has been most frequently adopted in the art, is the phosphomolybdate reduction methods, the representative thereof being the Fiske-Subbarow method. In the phosphomolybdate reduction method, phosphate ions are allowed to react with a molybdate to produce phosphomolybdic acid, and then reduced by a reducing agent to be converted to molybdenum blue which is colorimetrically analyzed to effect quantitative analysis of inorganic phosphorus. However, this known method has disadvantages such that deproteinization operation, which is conducted by the addition of trichloroacetic acid or like reagent, is required. Further, the color stability after the development of color is relatively poor to require strict time control from the color development to the colorimetrical measurement. In addition, the reducing agent used in the coloring reaction has poor storage stability. These disadvantages induce inconvenience when the method is used for a clinical test wherein a lot of samples must be processed rapidly. This known method has further problems that metallic parts of the instruments used for analysis operation are corroded by the use of a strong acid and that formed dye pigments tend to be adsorbed to the vessels or lines of the instrument. These problems pose hard obstacles for the realization of automated analyzing system for the quantitative analysis of inorganic phosphorus.
Particularly in the routine clinical tests in which a number of test samples are to be handled, it is demanded that the individual samples should be analyzed rapidly by simple operations, more desirably by automated operation sequence. To comply with the demand, dry analysis elements have been proposed (for example, by Unexamined Japanese Patent Publication Nos. 53888/1974 (corresponding to U.S. Pat. No. 3,992,158), 77356/1984 (corresponding to EP 0097952A) and 102388/1984 (corresponding to U.S. Pat. No. 4,861,552) and U.S. Pat. No. 4,459,358. Although it is desirous that the phosphomolybdate reduction method can be applied to such a dry analysis element, it is difficult to realize such an application.
In recent years, a few enzymatic analysis methods, by which inorganic phosphorus can be specifically analyzed without the need of deproteinization or similar pre-treatment, has been developed (Japan J. Clin. Chem., 11, 83, (1982), Unexamined Japanese Patent Publication Nos. 210998/1985 and 74499/1988). One of such methods is the PNP-XOD-POD method disclosed, for example, by Japan J. Clin. Chem., 11, 83, (1982), wherein inorganic phosphate Pi is allowed to react with inosine in the presence of purine nucleoside phosphorylase (hereinafter referred to as PNP) to produce hypoxanthine which is oxidized by the action of xanthine oxidase (hereinafter referred to as XOD) to form xanthine which is further oxidized to uric acid, and wherein hydrogen peroxide (H.sub.2 O.sub.2) produced at the oxidation step by the action of XOD is utilized for the color development of a color-former(dye precursor or chromogenic substrate) while using a peroxidase (hereinafter referred to as POD) and then the color density of the thus formed color is colorimetrically analyzed, the principle of this method being shown in FIG. 2. Since this method is a direct method wherein deproteinization is not required and is an enzymatic process in which no strong acid is used, there is no waste liquid problem otherwise arises in the conventional phosphomolybdate reduction method. This method is particularly convenient in that it is free from corrosion of automated analysis system and adsorption of formed dye pigment.
The PNP-XOD-POD method, as described above, may be applied to a dry analysis element. The dry analysis element is an analysis element including one or plural functional layers, at least one layer (or plural layers) of which contains an analytical reagent composition so as to form a coloring dye by the reaction taking place in the layer and the thus formed dye is colorimetrically analyzed by measuring the transmitting or reflecting light from the outside of the analysis element. Since such a dry analysis element is stored and preserved in the dry state prior to the analysis operation, there is no need of preparing the reagent at the measuring step. Furthermore, since the reagent has a higher stability in the dry state, the process using such a dry analysis element is improved in simplicity and speed of operation over the conventional wet process.
However, in the course of investigation for the realization of applying the aforementioned PNP-XOD-POD method to the dry analysis element, the inventors have found that there are problems in the storage stability and the measurable range.